Rapid polymer sequencer

ABSTRACT

Method and system for rapid and accurate determination of each of a sequence of unknown polymer components, such as nucleic acid components. A self-assembling monolayer of a selected substance is optionally provided on an interior surface of a pipette tip, and the interior surface is immersed in a selected liquid. A selected electrical field is impressed in a longitudinal direction, or in a transverse direction, in the tip region, a polymer sequence is passed through the tip region, and a change in an electrical current signal is measured as each polymer component passes through the tip region. Each of the measured changes in electrical current signals is compared with a database of reference electrical change signals, with each reference signal corresponding to an identified polymer component, to identify the unknown polymer component with a reference polymer component. The nanopore preferably has a pore inner diameter of no more than about 40 nm and is prepared by heating and pulling a very small section of a glass tubing.

ORIGIN OF THE INVENTION

The invention described herein was made, in part, by an employee of theUnited States Government and may be manufactured and used by or for theGovernment for governmental purposes without the payment of anyroyalties thereon or therefor.

TECHNICAL FIELD

The present invention is a method and system for rapidly and accuratelydetermining an ordered sequence of molecular units, such as bases in anucleic acid, such as DNA or RNA, and for fabricating a nanopore systemto facilitate the sequencing.

BACKGROUND OF THE INVENTION

Nanofabrication techniques offer the possibility to create solid statepores or apertures with diameters and lengths similar to diameters andlengths of single nucleotides or proteins. Solid state nanopores permituse of non-physiological conditions for structural manipulation ofbiopolymers, such as non-neutral pH levels, high temperatures and/orhigh voltage differences. Use of a solid state substrate will allow amore straightforward manipulation of surface chemistry in the pore,which may be critical to fine-tune the rate of nucleic acidtranslocation or the degree of ionic current reduction associated withpassage of a polymer, such as a poly-nucleotide through a nanopore.

Kasianowicz et al, in “Characterization of individual polynucleotidemolecules through a membrane channel,” Proc. Nat. Acad. Sci. vol. 93(1996) 195-223, have used a pore of diameter about 1.5 nanometers (nm)in the bacterial α-hemolysin ion channel protein, and have applied anelectrical field to drive a negatively charged polynucleotide throughthe pore from one side to the other, which transiently reduces ionicconductance through the pore. Akeson et al, in “Microsecond Time ScaleDiscrimination Among Polycytidylic Acis in Homopolymers or as SegmentsWithin Single RNA Molecules,” Biophys. Jour. Vol. 77 (1999) 3227-3233,have shown that polynucleotides of different lengths can bediscriminated by time duration of translocation as the nucleotidesequence passes through a pore. Translocation of different nucleotidehomopolymers reduces ionic conductance of α-hemolysin by characteristicamounts, which suggests that the individual nucleotides in aheteropolymer could be identified, if passed through a nanopore ofappropriate dimensions and composition. However, α-hemolysin has a porelength as long as a sequence of about 20 nucleotides so thatdiscrimination between individual nucleotides using α-hemolysin is notpossible.

What is needed is a system that provides rapid and accurateidentification of ordered components of a nucleic acid, protein orsimilar polymer, at rates up to and above one component per μsec.Preferably, the approach should adequately discriminate between thedifferent ordered components present in the polymer and provide accurateordering, with an acceptable error rate that is controllable by varyingthe rate at which the polymer components pass through and is read by thesystem.

SUMMARY OF THE INVENTION

These needs are met by the invention, which provides a system andassociated method that relies upon a pore at a pipette tip, having apore diameter as small as 1-40 nm, preferably containing a selectedalkali halide, ammonium compound (e.g., NH₄, N(CH₃)₄, or a suitableionic organic compound or ionic inorganic compound (e.g., CaSO₄,Mg_(m)(PO₄)_(n)). In one embodiment, a voltage difference is impressed,in a longitudinal direction or in a transverse direction, across anionic liquid within the pore, and a varying ionic current through thepore, or a varying electron current across the pore (referred tocollectively as an “electrical current”) is measured in response topassage of each of an ordered sequence of polymer components, such asnucleotides in a nucleic acid or proteins, through the pore.

In one embodiment, the method includes steps of:

providing a pipette having a longitudinal axis and having a taperedregion having a pore with a selected pore diameter in a range of 1-40nanometers (nm);

providing a selected liquid in contact with an interior surface of thepore;

impressing a selected voltage difference across the selected liquidwithin the pipette pore substantially parallel to the pipettelongitudinal axis direction, and providing an ionic current valueinduced in the selected liquid; and

passing an unknown polymer molecule, having a sequence of polymercomponents, through the pore, and determining a change in the ioniccurrent signal induced by passage of each of the polymer componentsthrough the pore. In another embodiment, the voltage difference isimpressed transversely, across the pore, and a transverse electroniccurrent, induced in response to passage of each of the polymercomponents through the pore, is measured.

In another embodiment, a method for producing the pore includes stepsof:

heating a hollow cylinder of a selected pipette material, having firstand second cylinder ends, having a longitudinal axis and having aselected initial inner diameter, with a selected heating source for atleast one of first and second longitudinal locations for at least one offirst and second selected time intervals;

translating one of the first and second cylinder ends relative to theother of the first and second cylinder ends during a selected third timeinterval that partly or wholly overlaps at least one of the first timeinterval and the second time interval; and

allowing the hollow cylinder to separate into at least first and secondpipettes and at least one of the first and second pipettes has a porewith a pore diameter in a range 1-40 nanometers (nm).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C illustrate apparati for practicing the invention.

FIGS. 2A, 2B and 2C graphical views of typical sequences of ionic orelectron current values measured with no polymer component present (2A),and in response to passage of a polymer component through a pore (2B,2C).

FIGS. 3, 4 and 5 are flow charts illustrating procedures for practicingthe invention according to two embodiments.

FIGS. 6A-6F graphically illustrate time variations that can be appliedto an impressed voltage difference used in the invention.

FIGS. 7A and 7B illustrate formation of a pipette tip for use in theinvention.

DESCRIPTION OF BEST MODES OF THE INVENTION

FIG. 1A illustrates one embodiment of apparatus for measurement oflongitudinal ionic current in practicing the invention. The system 11includes a glass pipette 12, having a longitudinal axis AA and having atapered tip region 13, with a pore 13 p having a selected pore minimuminner diameter d(min) in a preferred range of 1-40 nm, or larger ifnecessary. A length of the interior surface of the tip region 13 isprovided with a selected first liquid 14-1 including an ionizablemolecule including an alkali halide (NaCl, KCl, NaBr, KBr, MgCl₂, CaCl₂,MgClBr, etc.), ammonium compound (e.g., NH₄, N(CH₃)₄, or a suitableionic organic compound or ionic inorganic compound (e.g., CaSO₄,Mg_(m)(PO₄)_(n)). A portion of the pipette adjacent to the pore isimmersed in a selected second liquid 14-2, which may be, but need notbe, the same as the first liquid 14-1. The pore 13 p has a pore lengthL(pore) in a selected length range. A voltage difference ΔV, having avalue in a range 10-2000 milliVolts, is impressed substantially in thelongitudinal axis direction across the liquid in the pore, and an ioniccurrent value IC through the pore is measured by an electrical currentmeasurement module 15 The voltage difference may, for example, beprovided by a first electrode 17A, positioned within the first liquid14-1 in the interior surface of the pore 13 p, and a second electrode17B, positioned within a “bath” 19 of the second liquid 14-2 surroundingthe pore. One or both of the electrodes, 17A and 17B, may includeAg/AgCl or another substance known to provide reversible current and tohave low offset voltage in ionic solutions.

FIG. 1B, illustrating an embodiment for measuring transverse electroniccurrent, is similar to FIG. 1A, except that spaced apart electrodes, 18Aand 18B, replacing the electrodes 17A and 17B, are arranged on oradjacent to a perimeter of the pore 13 and an electronic current flowsfrom 18A to 18B in response to imposition of a voltage difference ΔVbetween these electrodes.

FIG. 1C illustrates a different configuration of the pore 13 p,according to the invention. In FIG. 1C, different portions of an end 12e of the tip substantially face each other and define an effective porelength L(pore) that is approximately equal to a thickness of the pipette12 at an end of the pipette. This configuration is preferred where thepore width d(min) is to be made as small as possible (e.g., less than orequal to 1.5 nm).

In one approach, an interior surface of the pore 13 p is left uncoatedin practicing the invention. Preferably, the interior surface of thepore 13 p is coated or wetted or otherwise provided with aself-assembling monolayer (“SAM”) 21 of a selected material that willmanifest hydrogen bonding, van der Waals interaction and/or similarreversible, transient interactions with a class of polymers of interest.The SAM substance provided on an interior surface of the pore 13 p maybe octadecyltrichlorosilane (C₁₈H₃₇SiCl₃ or “OTS”), as discussed bySagiv in “Organized Monolayers by Absorption. 1. Formation and Structureof Oleophobic Mixed Monolayers on Solid Surfaces,” J. Amer. Chem. Soc.vol. 102 (1980) pp. 92-98, or may be another suitable substance thatwill interact with a polymer components passing through the pore 13 pand allow measurement of a modulated ionic current signal or electroncurrent signal that is characteristic of a particular polymer component.Other SAM substances that may be used include alkylsiloxane monolayers,alkylsilanes, trimethoxysilanes, mono-, di- and tri-chlorosilanes,octadecylsilanes, organochlorosilanes, aminosilanes,perfluorodecyltrichlorosilanes and aminopropylethoxysilanes.

From another perspective, a stable SAM can be formed usingsulfur-containing absorbates on gold, chlorosilanes or alkoxysilanes onglass, and fatty acids on a metal oxide surface.

As used herein “SAM” includes an array of substantially identicalmolecules (e.g., containing a silane component) covalently attached to aglass surface and oriented substantially perpendicular to the surface,which interact, without permanent bonding, with a selected group of oneor more solution molecules that pass near the SAM array. A SAM may beused to provide transient interactions with a polymer ubit passingthrough a nanopore and/or may be used to tailor the effectivelongitudinal and/or transverse dimensions (diameter, etc.) of ananopore.

Formation of self-assembling monolayers on a gas or liquid interfacewere first reported by Langmuir in J. Am. Chem. Soc., vol. 39 (1917)1848, and were first shown to be formable on a solid surface byBlodgett, J. Am. Chem. Soc., vol. 57 (1935) 1007. Spontaneous formationof a SAM on a solid substrate was first demonstrated by Bigelow, Pickettand Zisman, J. Colloid Interface Sci., vol. 1 (1946) 513 Chechik, Crooksand Stirling, in “Reactions and Reactivity in Self-AssembledMonolayers,” Advanced Materials, 2000, 1161-1171, define a SAM as amonomolecular film of surfactant, formed spontaneously on a substrateupon exposure to a surfactant solution and provide a general review ofuse of SAMs. A SAM may be disordered or close packed, depending on thedegree of wetting of the substrate surface. As noted by Chechik et al,ibid, a SAM is ideally suited as a scaffold to graft a polymer ontosolid surfaces, because of the high density of functional groups,relatively small number of defects, and a well defined structure. Amolecule that is part of a SAM may be terminated by various functionalgroups, such as OH and amines.

A second example of an SAM is (16-Mercapto)hexadecanoic acid (MHA) on agold substrate, as studied and reported by J. Lahann et al in Science,vol. 239 (2003) pp 371-374. MHA includes hydrophobic chains capped byhydrophilic carboxylate end groups. Cleavage of the carboxylate endgroups provides a low density SAM of hydrophobic chains. Application ofa small electrical potential or voltage difference (e.g., −1054mV≦V≦+654 mV to the negatively charged carboxylate groups provides anattractive force that causes a conformation change in the hydrophobicchains, whereby an all-trans conformation becomes partly trans andpartly gauche conformation, with substantial qualitative andquantitative changes in associated sum-frequency generation (SFG)spectroscopic variations associated with the different conformations.The conformational changes are reversible so that removal of the appliedelectrical potential causes a return to the relatively featureless SFGspectroscopic variations associated with the original hydrophobic chainconformations (all trans). Viewed from another perspective, change inhydrophobic chain conformations associated with a specific change, suchas a variation in translocation associated with passage of differentpolymer units through a nanopore in which a very thin layer of MHA ongold is provided, would cause a measurable change in electrical currentor change in electrical potential (tens to hundreds of millivolts)associated with passage of each (different) polymer unit.

In the absence of passage of a polymer component through the pore, asteady ionic current through the pore (or electronic current across thepore) will develop in response to impressing a small voltage differencein a longitudinal (or transverse) direction, as shown graphically inFIG. 2A.

An ordered sequence of polymer components, such as a nucleic acid (e.g.,DNA or RNA) is passed through the pore 13 p, allowing development of asequence of changing ionic current values, as illustrated in FIGS. 2Band/or 2C. In FIG. 2B, passage of each polymer component through thepore 13 p is assumed to produce an approximately square wave signal,having an approximately constant characteristic amplitude for a smalltime interval that corresponds to the time required for that polymercomponent to pass through the tip. In FIG. 2C, passage of each polymerunit through the pore 13 p is assumed, more realistically, to produce asignal having a characteristic, time varying signal shape, acharacteristic average amplitude and a characteristic shape parameter,for a small time interval that corresponds to the time required for thatpolymer component to pass through the tip.

Where one or more polymer components passes through the pore,translocation will cause the steady ionic current shown in FIG. 2A tochange with time in response to passage of the polymer component throughthe pore and the accompanying translocation. If, for example, thepolymer sequence is a nucleic acid, such as DNA (alternatively, RNA),each nucleotide will contain one of the four bases adenine (A), cytosine(C), guanine (G) and thymine (T) (alternatively, adenine, cytosine,guanine and uracil (U) for RNA). Ideally, each of the four bases (forDNA or for RNA) will produce a distinguishable change in ionic currentsignal as that nucleotide passes through the pipette tip, as suggestedin FIG. 2B or FIG. 2C.

Under the influence of an applied voltage difference, negatively chargednucleotides or other polymer units are driven through the pore, and apolynucleotide strand can thus be threaded from one side of a lipidbiolayer to the other. A steady electrical current that is present inthe pore in the absence of a polymer unit is partly occluded duringtranslocation. In principle, polymer units of different lengths can bedistinguished from each other by translocation duration, and severalhomopolymers of different composition can be distinguished based oncharacteristic levels of electrical current reduction.

FIG. 3 is a flow chart of a procedure for practicing the invention. Instep 31 of FIG. 3, a pipette, having a longitudinal axis and having atapered tip with an associated pore having a selected pore minimum innerdiameter d in a preferred range (e.g., d=1-40 nm) is provided, and aselected self-assembling monolayer (SAM) is optionally provided on someportion of the pore surface. In step 32, a selected first liquidcontaining ions is provided in the interior surface of the pore,preferably containing an alkali halide, ammonium compounds (e.g., NH₄,N(CH₃)₄, or a suitable ionic organic compound or ionic inorganiccompound (e.g., CaSO₄, Mg_(m)(PO₄)_(n)). More generally, the selectedfirst liquid may be any solution that provides a concentration p of ionsat least equal to a threshold value ρ(ion;thr), for example,ρ(ion;thr)≧10^(x) cm⁻³. The liquid may include the polynucleotide orother polymer that is to be identified. In step 33, a voltage differencehaving a value in a range ΔV=10-2000 milliVolts is impressed on theliquid in the pore, in a direction substantially parallel to the pipettelongitudinal axis. If the polymer has a net electrical charge, thepolarity of the voltage difference is chosen to induce the polymer topass through (or across) the pore. In step 34, ordered components in apolymer (unknown) are sequentially passed through the pore, and each ofa sequence of changes in ionic current signals is measured, resulting ina sequence of measured values such as the sequences shown in FIG. 2B orFIG. 2C.

In step 35 (optional), the sequence of changes in measured ionic currentsignals CIC(t;meas) is compared, one-by-one or in consecutive groups,with reference change signals CIC(t;ref;n), numbered n=1, . . . , N(N≧2) in a reference signal database. Each reference change signalcorresponds to a reference polymer component. In step 36 (optional),each polymer component (e.g., a nucleotide containing a particular base)in the unknown sequence is assigned to the reference polymer componenthaving a reference ionic current change signal that is most similar, insome quantitative sense, to the measured (changes in) ionic currentchange signal. Optionally, steps 35 and 36 are performed off-line

The signal comparison step 35 is optionally implemented as follows. Theionic current change signal CIC(t;meas) for the unknown polymer sequenceis measured at a sequence of time values t_(m), producing a sequence ofmeasured ionic current change values {CIC(t_(m);meas)}_(m). (m=1, . . ., M; M≧2) This sequence of measured ionic current change values iscompared with a reference sequence (n) of ionic current change signalvalues {CIC(t_(m)+τ(n);ref;n)}_(m), where τ(n) is a selected time shiftthat may vary with the reference number n being considered, by computingan error valueMε(n)={w _(m) |CIC(t _(m);meas)−CIC(t _(m)+τ(n);ref;n)|^(p)}^(1/p),  (1)m=1where {w_(m)}_(m) is a selected sequence of non-negative weight values(at least one positive) and p is a selected positive number (e.g., p=1,1.6 or 2). Reference ionic current change signals CIC(t_(m)+τ;ref;n) forwhich the error satisfies ε(n)>ε(thr), where ε(thr) is a selectedpositive threshold value, are discarded and not considered further forthis measured ionic current change value sequence {CIC(t_(m);meas)}_(m).When at least one error value satisfies ε(n)≦ε(thr), the “survivingcollection”SC={CIC(t _(m)+τ(n);ref;n)|ε(n)≦ε(thr)}  (2)of all reference signals with error values that satisfy the inequalityε(n)≦ε(thr), are considered, and the reference ionic current changesignal CIC(t_(m)+τ(n);ref;n) that provides the smallest error ε(n) isassigned to the unknown polymer unit. When the surviving collection SCis an empty set, because no error value satisfies ε(n)≦ε(thr), thesystem assigns a selected symbol, such as UNK, to this polymer unit.

The comparison procedure can be summarized in a flow chart in FIG. 4. Instep 41, the error ε(n), defined as in Eq. (1) or in another suitablemanner, for each reference change signal CIC(t_(m)+τ(n);ref;n) in thedatabase is computed. In step 42, the surviving collection SC ofreference signals is determined. In step 43, the system determines if SCis an empty set. If the answer to the query in step 43 is “yes,” thesystem assigns a special symbol (e.g., UNK) to the correspondingmeasured ionic current value in step 44, indicating that no referencechange signal IC(t_(m)+τ(n);ref;n) is sufficiently similar to themeasured ionic current change signal. If the answer to the query in step43 is “no” (SC is non-empty), the system identifies, in step 45, eachreference change signal CIC(t_(m)+τ(n);ref;n=n′) for which thecorresponding error satisfiesε(n′)=min_(1≦n≦N)ε(n).  (3)In step 46, the system identifies at least one reference polymercomponent, for which the corresponding reference change signalCIC(t_(m)+τ(n′);ref;n′) is in the surviving collection SC, with theunknown polymer component whose signal was measured.

FIG. 5 is a flow chart of an alternate procedure for practicing theinvention, using a transverse voltage difference. In step 51 of FIG. 5,a pipette, having a longitudinal axis and having a tapered tip with anassociated pore having a selected pore minimum inner diameter d in apreferred range (e.g., d=1-40 nm) is provided, where a selectedself-assembling monolayer is optionally provided on an interior surfaceof the pore. In step 52, the pore interior is provided with a selectedfirst liquid, preferably containing an alkali halide, ammonium compounds(e.g., NH₄, N(CH₃)₄, or a suitable ionic organic compound or ionicinorganic compound (e.g., CaSO₄, Mg_(m)(PO₄)_(n)), so that the firstliquid is present within the pore. More generally, the selected liquidmay be any solution that provides at least a concentration ρ ofelectrons at least equal to a threshold value ρ(ion;thr), for example,ρ(ion;thr)≧10^(x) cm⁻³. In step 53, a voltage difference having a valuein a range ΔV=10-2000 milliVolts, or more if desired, is impressed onthe first liquid in the pore, in a direction substantially transverse tothe pipette longitudinal axis. In step 54, a polymer sequence (unknown)is sequentially passed through the pore, and each of a sequence ofelectron current change signals is measured, resulting in a sequence ofmeasured values such as the sequences shown in FIG. 2B or FIG. 2C. Theelectron signals resulting from imposition of the transverse voltagedifference are likely to be different from the corresponding ioniccurrent signals resulting from imposition of a longitudinal voltagedifference.

In step 55 (optional), the sequence of measured electron current changesignals CEC(t_(m);meas) is compared, one-by-one or in consecutivegroups, with reference change signals CEC(t_(m)+τ(n);ref;n), numberedn=1, . . . , N′ (N′≧2) in a reference signal database. In step 56(optional), each polymer unit (e.g., a nucleotide containing aparticular base) in the unknown sequence is assigned to the referencepolymer component having a reference electron current value that is mostsimilar to the measured electron current signal. Step 55 may, forexample, be implemented by analogy with implementation of step 35 inFIG. 3, with electronic change signals, CEC(t_(m);meas) andCEC(t_(m)+τ(n);ref;n) replacing the corresponding ionic current changesignals in Eqs. (1) and (2).

The voltage difference amplitude ΔV(t), impressed longitudinally ortransversely across the selected liquid, may be substantially uniform intime, as illustrated in FIG. 6A, may be substantially monotonicallyincreasing in time (FIG. 6B), may be substantially monotonicallydecreasing in time (FIG. 6C), may be substantially a step function intime (FIG. 6D), may vary substantially sinusoidally in time (FIG. 6E),may vary substantially trapezoidally in time (FIG. 6F), with temporallength segments τ1, τ2 and τ3, or may have another suitable time varyingshape. The trapezoidal variation shown in FIG. 6F includes a triangularvariation, in which the middle segment has length τ2=0.

A tip region of a pipette (quartz glass, aluminosilicate glass,borosilicate glass or other suitable glass) having an appropriateminimum inner diameter may be formed using the following procedure,illustrated in FIGS. 7A and 7B. A selected middle region 73, having apreferred length LH in a range 0.1-2 cm or more, of a pipette 71 with ahollow core is heated or otherwise receives substantial thermal energy,using a laser, infrared source or a heated metal filament 75 and(optional) associated focusing system 76, at one or more locations,x=x1, x=x2, etc., for one, two or more time intervals, of length Δt1,Δt2, etc. The time intervals may partly or wholly overlap or may beisolated from each other. As the heating or irradiation continues, oneor both of first and second ends, 77-1 and 77-2, of the pipette ispulled with a selected force F, optionally 10¹-10⁷ dynes or more, or ata selected displacement rate, v of a few mm/sec, so that the first andsecond ends are displaced relative to each other. The pipette 71separates into two pipette segments, 71-1 and 71-2, in the (last)heating cycle, and at least one of the two resulting pipette segmentshas a hollow core (a pore) with a pore minimum inner diameter d(min).Tip parameters (thickness, nanopore diameter, nanopore length, etc.) canbe partly controlled by appropriate choice of one or more of theparameters heating rate, LH, Δt(irr), F and/or v.

Suitable applications of the invention, using ionic current orelectronic current, include the following: (1) counting of genomic ornon-genomic fragments, by identification of a first end and/or a secondend of each fragment that passes through a nanopore; (2) identificationof locations of single strand segments and double strand segments in a“mixed” DNA sequence passing through a nanopore; (3) discriminationbetween single strands and double strands of DNA passing through ananopore; and (4) identification of individual nucleotides in singlestrand DNA passing through a nanopore; (5) identification ofcorresponding base pairs (e.g., cytosine-guanine, adenine-thymine andadenine-uracil) in a double strand DNA or RNA passing through ananopore, and (6) estimation of polymer component length by correlationwith length of time interval for translocation.

What is claimed is:
 1. A method of fabricating a nanopore, the methodcomprising: heating a hollow cylinder of a pipette material, comprisingprimarily at least one of quartz glass, aluminosilicate glass andborosilicate glass, by a process comprising use of at least one of alaser, an infrared light source and a heated metal for heating one ormore locations on the cylinder for a first time interval, the hollowcylinder having first and second cylinder ends, having a longitudinalaxis and having a non-zero initial inner diameter; and applying amachine controlled translation force to translate at least one of thefirst and second cylinder ends relative to the other of the first andsecond cylinder ends by a change in end-to-end separation distance nogreater than about 2 cm during a second time interval that partly orwholly overlaps the first time interval, in order to encourage thehollow cylinder to separate into at least first and second pipettes,each with a corresponding nanopore, with at least one pore diameter in arange of 1-40 nanometers (nm) and with at least one pore length nogreater than about 2 cm.
 2. The method of 1, further comprising choosingsaid heating source from a group of heating sources consisting of alaser, an infrared light source, and a heated metal.
 3. The method ofclaim 1, further comprising: providing a selected liquid in contact withan interior surface of said pore; impressing a non-zero voltagedifference across the selected liquid within said pore approximatelyparallel to a longitudinal axis direction of said cylinder, anddetermining at least one of an electrical current value and an ioniccurrent value induced in the selected liquid; and passing a polymermolecule, having a sequence of polymer components, through said pore ina first direction, determined with reference to the longitudinal axisdirection, and determining at least one of an electrical current signaland an ionic current signal induced by passage of each of the polymercomponents through said pore.
 4. The method of claim 3, furthercomprising selecting material for said pipette from a group of materialsincluding quartz glass, aluminosilicate glass and borosilicate glass. 5.The method of claim 3, further comprising passing said polymer sequencethrough said pore at an average rate in a range of 1-1000 polymercomponents per msec.
 6. The method of claim 3, further comprisingchoosing said polymer sequence to be a nucleic acid sequence includingthe bases adenine, cytosine and guanine and at least one of the basesthymine and uracil.
 7. The method of claim 3, further comprisingselecting said voltage difference from a group of time-dependentdifferences including a difference that (i) is approximately uniform intime; (ii) increases monotonically with time; (iii) decreasesmonotonically with time; (iv) is a step function in time; (v) variessinusoidally with time; and (vi) varies trapezoidally with time.
 8. Themethod of claim 3, further comprising choosing said selected liquid toinclude at least one of an alkali halide, an ammonium compound, an ionicorganic compound and an ionic inorganic compound.
 9. The method of claim1, further comprising providing a self-assembling monolayer of aselected substance on a selected portion of said interior surface ofsaid pore.
 10. The method of claim 9, further comprising choosing saidself assembling monolayer to include at least one of: (i)octadecyltrichlorosilane on glass and (ii) (16-Mercapto)hexadecanoicacid on a gold substrate.
 11. The method of claim 1, further comprising:providing a selected liquid in contact with an interior surface of saidpore; impressing a non-zero voltage difference across the selectedliquid within said pore transverse to a longitudinal axis direction ofsaid cylinder, and determining an ionic current value induced in theselected liquid; and passing a polymer molecule, having a sequence ofpolymer components, through said pore in a first direction, determinedwith reference to the longitudinal axis direction, and determining anionic current signal induced by passage of each of the polymercomponents through said pore.
 12. The method of claim 11, furthercomprising passing said polymer sequence through said pore at an averagerate in a range of 1-1000 polymer components per msec.
 13. The method ofclaim 11, further comprising choosing said polymer sequence to be anucleic acid sequence including the bases adenine, cytosine and guanineand at least one of the bases thymine and uracil.
 14. The method ofclaim 11, further comprising selecting said voltage difference from agroup of time-dependent differences including a difference that (i) issubstantially uniform in time; (ii) increases monotonically with time;(iii) decreases monotonically with time; (iv) is a step function intime; (v) varies sinusoidally with time; and (vi) varies trapezoidallywith time.
 15. The method of claim 11, further comprising choosing saidselected liquid to include at least one of an alkali halide, an ammoniumcompound, an ionic organic compound and an ionic inorganic compound. 16.The method of claim 1, further comprising providing a self-assemblingmonolayer of a selected substance on a portion of said interior surfaceof said pore.
 17. The method of claim 16, further comprising choosingsaid self assembling monolayer to include at least one of: (i)octadecyltrichlorosilane on glass and (ii) (16-Mercapto)hexadecanoicacid on a gold substrate.
 18. The method of claim 1, further comprisingapplying said machine controlled translation force in a range of between10 dynes and 10 million dynes in a direction corresponding to saidlongitudinal axis.